Figure 4.
Figure 4. Silencing of CXCL1 prevents the hyperexcitability of hippocampal neurons in endothelial Cdk5-deficient mice. (A) Representative immunostaining images and quantification of GFAP (green) and S100β (red) in primary cultured hippocampal astrocytes from Cdk5f/f mice incubated with PBS and recombinant CXCL1 for 6 h (20 ng; n = 5 mice per group; ***, P < 0.001; unpaired two-tailed Student’s t test). Bar, 100 µm. (B) Glutamate uptake by primary cultured hippocampal astrocytes in the recombinant CXCL1 6-h incubation group and the control group was assessed after puff application of 100 µM glutamate for 500 ms before and after the application of 200 µM DHK (n = 5 mice per group; ***, P < 0.001; unpaired two-tailed Student’s t test). (C) Schematic representation of AAV-BR1 constructs indicating the inverted terminal repeats (ITR) at both ends and CMV promoter-driven EGFP (BR1-Con) or CMV promoter-driven shCxcl1 with EGFP (BR1-shCxcl1). (D) The relative mRNA level of Cxcl1 in primary cultured ECs from the cerebral microvessels was evaluated in BR1-Con– and BR1-shCxcl1–injected Cdk5f/f mice at 4 wk (n = 3 mice per group; *, P < 0.05; unpaired two-tailed Student’s t test). (E) Representative confocal images and quantification of GFAP (green) and S100β (red) in Cdk5f/f and Cdh5-Cre;Cdk5f/f mice injected with BR1-Con and BR1-shCxcl1 (n = 3 mice per group; **, P < 0.01; ***, P < 0.001; one-way ANOVA followed by Tukey’s multiple comparisons test). Bars: main images, 100 µm; insets, 50 µm. (F) PTZ-induced seizure onset latency and duration in Cdk5f/f and Cdh5-cre;Cdk5f/f mice injected with BR1-Con and BR1-shCxcl1 at 4 wk (n = 5 mice per group; *, P < 0.05; **, P < 0.01; one-way ANOVA followed by Tukey’s multiple comparisons test). (G) Traces showing sEPSC and mEPSC recorded in BR1-Con- and BR1-shCxcl1-injected mice at 4 wk. (H–K) Cumulative probability plots summarizing the mean sEPSC amplitudes (H) and sEPSC inter-event intervals (I) and cumulative probability plots summarizing the mean mEPSC amplitudes (J) and mEPSC inter-event intervals (K) in Cdh5-Cre;Cdk5f/f and Cdk5f/f mice injected with BR1-Con and BR1-shCxcl1 at 4 wk. The insets depict the average sEPSC and mEPSC amplitudes and frequencies (n = 3–5 mice per group; *, P < 0,05; **, P < 0.01; ***, P < 0.001; unpaired two-tailed Student’s t test). The data are presented as means ± SEM. (L) Representative GLT1 transport currents following DHK incubation in the hippocampus from 4-wk-old Cdh5-Cre;Cdk5f/f and Cdk5f/f mice with BR1-Con and BR1-shCxcl1 injection. Quantification of the GLT1 transport currents is shown on the right (n = 3–5 mice per group; **, P < 0.01; ***, P < 0.001; one-way ANOVA followed by Tukey’s multiple comparisons test). The numbers inside the bars represent the numbers of cells from three to five mice. The error bars represent means ± SEM; n.s., not significant.

Silencing of CXCL1 prevents the hyperexcitability of hippocampal neurons in endothelial Cdk5-deficient mice. (A) Representative immunostaining images and quantification of GFAP (green) and S100β (red) in primary cultured hippocampal astrocytes from Cdk5f/f mice incubated with PBS and recombinant CXCL1 for 6 h (20 ng; n = 5 mice per group; ***, P < 0.001; unpaired two-tailed Student’s t test). Bar, 100 µm. (B) Glutamate uptake by primary cultured hippocampal astrocytes in the recombinant CXCL1 6-h incubation group and the control group was assessed after puff application of 100 µM glutamate for 500 ms before and after the application of 200 µM DHK (n = 5 mice per group; ***, P < 0.001; unpaired two-tailed Student’s t test). (C) Schematic representation of AAV-BR1 constructs indicating the inverted terminal repeats (ITR) at both ends and CMV promoter-driven EGFP (BR1-Con) or CMV promoter-driven shCxcl1 with EGFP (BR1-shCxcl1). (D) The relative mRNA level of Cxcl1 in primary cultured ECs from the cerebral microvessels was evaluated in BR1-Con– and BR1-shCxcl1–injected Cdk5f/f mice at 4 wk (n = 3 mice per group; *, P < 0.05; unpaired two-tailed Student’s t test). (E) Representative confocal images and quantification of GFAP (green) and S100β (red) in Cdk5f/f and Cdh5-Cre;Cdk5f/f mice injected with BR1-Con and BR1-shCxcl1 (n = 3 mice per group; **, P < 0.01; ***, P < 0.001; one-way ANOVA followed by Tukey’s multiple comparisons test). Bars: main images, 100 µm; insets, 50 µm. (F) PTZ-induced seizure onset latency and duration in Cdk5f/f and Cdh5-cre;Cdk5f/f mice injected with BR1-Con and BR1-shCxcl1 at 4 wk (n = 5 mice per group; *, P < 0.05; **, P < 0.01; one-way ANOVA followed by Tukey’s multiple comparisons test). (G) Traces showing sEPSC and mEPSC recorded in BR1-Con- and BR1-shCxcl1-injected mice at 4 wk. (H–K) Cumulative probability plots summarizing the mean sEPSC amplitudes (H) and sEPSC inter-event intervals (I) and cumulative probability plots summarizing the mean mEPSC amplitudes (J) and mEPSC inter-event intervals (K) in Cdh5-Cre;Cdk5f/f and Cdk5f/f mice injected with BR1-Con and BR1-shCxcl1 at 4 wk. The insets depict the average sEPSC and mEPSC amplitudes and frequencies (n = 3–5 mice per group; *, P < 0,05; **, P < 0.01; ***, P < 0.001; unpaired two-tailed Student’s t test). The data are presented as means ± SEM. (L) Representative GLT1 transport currents following DHK incubation in the hippocampus from 4-wk-old Cdh5-Cre;Cdk5f/f and Cdk5f/f mice with BR1-Con and BR1-shCxcl1 injection. Quantification of the GLT1 transport currents is shown on the right (n = 3–5 mice per group; **, P < 0.01; ***, P < 0.001; one-way ANOVA followed by Tukey’s multiple comparisons test). The numbers inside the bars represent the numbers of cells from three to five mice. The error bars represent means ± SEM; n.s., not significant.

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